PROJECT DESCRIPTION Cells critically regulate their volume in response to hypo-osmotic swelling by transporting chloride and small organic osmolytes out of the cell through so-called volume-regulated anion channels (VRACs). Native VRACs have been studied extensively using electrophysiological techniques for nearly three decades. They are ubiquitously expressed in mammalian cells and have been implicated in diverse functions, in addition to cell volume regulation, such as cell proliferation, cell migration, regulation of endothelial cell calcium signaling, release of excitatory amino acids in the brain, and regulation of insulin secretion from pancreatic beta cells. However, many of these studies suffered from non-specific pharmacological tools that could have confounding effects on unrelated targets and pathways. For example, our lab has recently discovered that DCPIB, the best- in-class inhibitor of VRACs, inhibits mitochondrial respiration, independently of VRAC function. The five genes encoding VRACs were recently identified in two independent genome-wide siRNA screens and are named LRRC8 (for Leucine Rich Repeat Containing) A-E. These discoveries create unprecedented opportunities for studying the molecular physiology of these important channels. The overarching goal of this proposal is to employ leading-edge techniques in electrophysiology, site-directed mutagenesis, high-throughput fluorescence assays, and medicinal chemistry to develop the molecular pharmacology of VRAC channels. In Specific Aim 1, I will employ fluorescence quenching assays and patch clamp electrophysiology to determine if VRACs containing different LRRC8 subunits can be distinguished pharmacologically. I will generate the specific VRAC subtypes, by heterologously expressing LRRC8A plus one other subunit (i.e. LRRC8C, LRRC8D, or LRRCDE) using an HCT116 cell line in which all five LRRC8 subunits have been knocked out with CRISPR (i.e. HCT116- LRRC8-/- cells). In Specific Aim 2, I will use site-directed mutagenesis to introduce cysteine residues in transmembrane domains 1-4 of LRRC8A to determine if DCPIB is a pore blocker of VRAC. To minimize the contribution of subunit stoichiometry variability in these experiments, I will use a newly developed monomeric chimera of LRRC8A containing the first extracellular loop (EL1) of LRRC8C that forms volume-regulated anion channels whose properties are virtually identical to that of the wild type channel. DCPIB block of mutagenized channels will be determined by fluorescence quenching assays and patch clamp electrophysiology. In Specific Aim 3, I will employ medicinal chemistry, fluorescence quenching assays, and patch clamp electrophysiology to develop DCPIB analogs that inhibit VRAC but do not inhibit mitochondrial function, as confirmed by performing Seahorse XF Cell Mito Stress tests. These studies represent the first efforts to define the molecular pharmacology of cloned VRAC channels and will create important new insights and tools for probing the structure-function relationships, physiology, and therapeutic potential of VRACs in metabolic diseases.